nonviral technologies for gene therapy in cardiovascular research

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REVIEW ARTICLE

NONVIRAL TECHNOLOGIES FOR GENE THERAPY IN CARDIOVASCULAR RESEARCH Cheng-Huang Su1,2*, Hung-I Yeh1,2,3, Charles Jia-Yin Hou1, Cheng-Ho Tsai1,2,3 1

Division of Cardiology, Department of Internal Medicine, Mackay Memorial Hospital, 2 Mackay Medicine, Nursing and Management College, and 3Taipei Medical University, Taipei, Taiwan.

SUMMARY Gene therapy, which is still at an experimental stage, is a technique that attempts to correct or prevent a disease by delivering genes into an individual’s cells and tissues. In gene delivery, a vector is a vehicle for transferring genetic material into cells and tissues. Synthetic vectors are considered to be prerequisites for gene delivery, because viral vectors have fundamental problems in relation to safety issues as well as large-scale production. Among the physical approaches, ultrasound with its associated bioeffects such as acoustic cavitation, especially inertial cavitation, can increase the permeability of cell membranes to macromolecules such as plasmid DNA. Microbubbles or ultrasound contrast agents lower the threshold for cavitation by ultrasound energy. Furthermore, ultrasound-enhanced gene delivery using polymers or other nonviral vectors may hold much promise for the future but is currently at the preclinical stage. We all know aging is cruel and inevitable. Currently, among the promising areas for gene therapy in acquired diseases, the incidences of cancer and ischemic cardiovascular diseases are strongly correlated with the aging process. As a result, gene therapy technology may play important roles in these diseases in the future. This brief review focuses on understanding the barriers to gene transfer as well as describing the useful nonviral vectors or tools that are applied to gene delivery and introducing feasible models in terms of ultrasound-based gene delivery. [International Journal of Gerontology 2008; 2(2): 35–47] Key Words: cavitation, gene therapy, microbubble, transfection efficiency, ultrasound, vector

Introduction Gene therapy is a novel approach that can be applied to any clinical therapeutic procedure in which nucleic acids are introduced into cells for the purpose of treating, curing or ultimately preventing disease1. Most commonly, the nucleic acids involved are DNA molecules that encode wild-type or modified gene products or

*Correspondence to: Dr Cheng-Huang Su, Division of Cardiology, Department of Internal Medicine, Mackay Memorial Hospital, 92, Section 2, ChungSan North Road, Taipei 10449, Taiwan. E-mail: [email protected] Accepted: April 22, 2008

International Journal of Gerontology | June 2008 | Vol 2 | No 2 © 2008 Elsevier.

proteins. The method used to transfer the nucleic acids into cells or tissues and the subsequent overexpression of the encoded proteins result in the therapeutic effects. Gene therapy can be targeted to germline (egg and sperm) or somatic (body) cells. In germline gene therapy, the parent’s eggs or sperms are changed with the goal of passing on the changes to their offspring. Germline gene therapy is not being actively investigated, at least not in large mammals or humans. In somatic gene therapy, alterations in the genetic makeup of individual somatic cells are not passed on to the next generation. Currently, gene therapy is solely concerned with delivering genes into somatic cells and does not involve genetic modification of the human germline, because this is not acceptable in most countries. 35

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In Taiwan, the development of gene delivery technologies is still in the very early stage. Applications of gene therapy require a Good Manufacturing Practice certificate by experienced researchers in medical centers, according to the guidelines from the Department of Health (www.doh.gov.tw/EN2006/index_EN.aspx), and need to fulfill the guidelines in terms of ethical and safety issues. For instance, applications of gene therapy are only allowed to involve somatic cells because of ethical problems. In addition, the possible lethal responses of humans to viral vectors (gene carriers) mean that carrier manipulation needs to be performed in an appropriate laboratory, such as a P1 laboratory for adeno-associated virus carriers, a P2 laboratory for adenovirus carriers or a P3 laboratory for retrovirus carriers. However, these advanced laboratories are solely located in medical centers or advanced research institutes in Taiwan. Consequently, regarding efforts to perform gene therapy in the future with respect to safety in local hospitals, gene therapists need to choose appropriate vectors, such as nonviral vectors. Cost-benefit analyses of gene therapy, i.e., analyses of the possibility of causing adverse effects/expense and gaining positive clinical effects, cannot be overemphasized before each treatment. The central focus of this brief review is the use of nonviral technologies and physical approaches, especially ultrasound (US)-based gene delivery, as potential tools for clinical gene delivery. Most of the basic technical principles regarding US are located in the section entitled “US-based Technologies in Gene Delivery”, and clinicians can therefore choose their own areas of interest.

immunodeficiency3. Although the gene therapy ended 2 years later, the integrated vector and adenosine deaminase gene expression in the T-cells persisted. Since then, more than 1,000 clinical trials have taken place worldwide. The diseases most commonly treated with gene therapy are cancers (66.5%), cardiovascular diseases (9%), monogenic diseases (8.2%), and infectious diseases (6.6%)4.

Candidate Diseases and Target Therapeutic Genes There are several promising areas for gene therapy in genetic and acquired diseases. For acquired diseases, cancers and cardiovascular diseases (more specifically, peripheral artery occlusive disease [a group of diseases caused by obstruction of peripheral arteries, mainly resulting from atherosclerosis]5–7, restenosis8,9, in-stent restenosis10,11, bypass graft failure12 and therapeutic angiogenesis for myocardial ischemia13–15) are the most explored. Among diseases related to the aging process, such as peripheral artery occlusive disease and ischemic heart disease, gene therapy may provide alternative roles. Regarding monogenic diseases, hemophilia, cystic fibrosis and familial hypercholesterolemia are of importance. The identification of defective genes in individual conditions with a view to introducing the normal counterparts by gene therapy is a major field of ongoing research.

Useful Vectors and General Approaches in Gene Therapy Overview of Applications in Gene Therapy The first investigation of vascular gene transfer was reported by Nabel et al.2, who transfected porcine endothelial cells (ECs) ex vivo with a retrovirus encoding the β-galactosidase gene and reintroduced the cells into the denuded iliofemoral artery of syngeneic pigs. Arterial segments isolated after 2 to 4 weeks contained ECs expressing β-galactosidase, thereby indicating successful incorporation of the transgene into the transduced cells. The first federally approved clinical trial of somatic gene therapy for a genetic disorder was started in the United States in September 1990. In this trial, the adenosine deaminase gene was transferred into the T-cells of two children with severe combined 36

Gene therapy is still in the early stages of development and remains predominantly experimental. Many factors have prevented researchers from developing successful gene therapy techniques. The process of gene delivery into cells for expression is known as transfection. Strictly speaking, viral vectors deliver exogenous nucleic acids by transduction, but for ease of use the term, transfection is used for all techniques in gene delivery. Successful transfection relies on achieving a biological balance between causing excessive damage to the cell and gaining adequate access of the DNA into the cytoplasm or nucleus. The first issue that needs to be addressed is the gene delivery tool. Gene delivery is achieved via vehicles designated vectors, which

International Journal of Gerontology | June 2008 | Vol 2 | No 2

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Nonviral Technologies for Gene Therapy

deliver therapeutic genes to the patients’ target cells or tissues. The three main categories of techniques that have been used to deliver genes to cells or tissues in gene therapy protocols are viral vectors (66.9%), nonviral vectors (26.6%), and physical delivery systems (1%)4. Currently, the most frequently used vectors are viruses16, of which the three most common are adenovirus, retrovirus, and adeno-associated virus. Owing to their highly evolved and specialized components, viral systems are by far the most effective means of DNA delivery, and they achieve high levels of both transfection efficiency (i.e., percentage of cells exposed to the vector that express the transgene) and transgene expression in the transfected cells. Scientists have tried to take advantage of virus biology and manipulate viral genomes to replace nonessential genes, particularly those necessary for viral replication, with therapeutic genes. However, despite their efficiency, viral vectors introduce other problems to the body, such as toxicity and the induction of immune and inflammatory responses17. Nonviral vectors have been developed to overcome some of these hurdles encountered with viral vectors, particularly the immunogenicity18. However, gene expression following nonviral transfection is often transient and tends to decrease rapidly within the first few days and disappear within 1 week. To date, some important nonviral alternatives that have been considered for gene delivery are complexes of DNA with lipids or polymers. In terms of physical nonviral delivery systems, needle-free injection, electroporation and US are the three major technologies currently under evaluation. Once a vector has been designed, two general approaches are used for somatic gene transfer: (1) the ex vivo model, in which cells are isolated, genetically modified and transplanted back into the same subject; and (2) the in vivo model, in which genes are administered directly to target cells in the body1.

Challenges in Gene Therapy The death of an 18-year-old boy, Jesse Gelsinger, during a gene therapy clinical trial in 1999 raised critical questions concerning the safety of experimental gene therapy trials19. Jesse suffered from a deficiency of ornithine transcarbamylase, a genetic defect that prevents the correct metabolism of ammonia, and died of complications from an inflammatory response shortly after receiving a dose of an adenovirus carrying a corrective


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gene. His death illustrates the challenges in gene therapy well and has given rise to much demanded discussion regarding the validity of using gene delivery vectors, especially viral vectors, and the evaluation of possible adverse effects in animal models.

Current Gene-transfer Systems A feasible gene therapy vector needs to meet the following three important criteria: safety; adequate gene transfer efficiency; and stable and reliable expression of the transgene (gene of interest) for an appropriate duration for the disease under treatment. There are at least five barriers that need to be overcome for successful gene delivery, namely cell entry, endosome escape, cytoplasmic transport and nuclear entry, in vitro and in vivo stability, and biocompatibility. Unfortunately, such ideal gene delivery systems are still under investigation. In the present section, nonviral vectors and physical methods are briefly introduced. It is well known that nonviral vectors are associated with low transfection efficiency, especially in vivo, and more transient expression in gene delivery. However, comparisons between them are not possible, because no reports have been published in this regard. The important nonviral vectors and physical approaches are summarized in Table 1 in terms of their key mechanisms.

Nonviral Vectors The safety concerns associated with viral vectors have encouraged the development of nonviral vectors. Plasmid DNA (pDNA) delivered by nonviral methods is not integrated into the cellular genome but maintained at an extrachromosomal site20. The most popular materials used in current nonviral applications include purified pDNA, lipids (usually a mixture of cationic and neutral lipids), and synthetic polymers.

Naked DNA The simplest nonviral gene delivery system currently in use in vivo is direct injection of naked pDNA. The use of naked pDNA without any carrier vehicle is also the safest method. However, owing to the rapid degradation of naked pDNA by nucleases in the serum and its clearance by the mononuclear phagocyte system in the systemic circulation, the expression levels after 37

■ Table 1.

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C.H. Su et al

Summary of the mechanisms for important nonviral vectors and physical approaches in gene delivery

Nonviral vector

Central mechanism

pDNA

Endocytosis

Lipid-based vectors

Endocytosis; condensation

Synthetic polymers (PEI, PLL)

PLL: endocytosis, condensation PEI: endocytosis, condensation, proton sponge

Physical approach

Central mechanism in gene delivery

Needle-free injection

Gene gun: high-pressure helium stream Jet gun: high-pressure solution

Electroporation

Electric pulse-induced promotion of cell membrane permeability

Ultrasound

Ultrasound energy-induced promotion of cell membrane permeability (sonoporation)

pDNA = plasmid DNA; PEI = polyethyleneimine; PLL = poly-L-lysine.

injection of naked DNA are generally limited. Although this technique has a low delivery efficiency, it is simple and safe with a very low risk of insertional mutagenesis. One of the promising approaches in this field is the combined use of naked DNA and a physical approach (such as electroporation) to enhance plasmid-mediated gene expression in muscle21–24.

Lipid-based vectors Lipid-based gene delivery, which was first reported in 1987 by Felgner et al.25, is still one of the major systems for increasing the transfection efficiency of naked DNA. Naked DNA forms liposomes or lipoplexes with positively charged lipids and detergents26,27, and these positively charged lipid–DNA complexes are capable of condensing with negatively charged DNA. Zabner et al.28 reported that condensed lipid–DNA complexes appear to be 100 nm or larger, at least in one dimension. Cationic lipids and cationic polymers both share this important property of complex formation with DNA. Furthermore, the resulting net positive charge of lipid–DNA complexes may facilitate fusion with the negatively charged cell membrane. Endocytosis is considered to be the major mechanism for liposomes to pass through the cell membrane28–30. Most of the liposome– DNA complexes are intracellularly degraded by lysosomal enzymes, and only 1% of the DNA enters the nucleus where it remains extrachromosomal. Therefore, transgene expression using liposomes is transient. Liposomes are nonpathogenic with no size limit for the transgene and are relatively cheap and easy to produce compared with viral vectors. Although the major limitation to their 38

application is the poor efficiency for transfecting nonproliferating cells, several studies have shown high levels of transgene expression following direct administration or injection31–33.

Synthetic polymers Synthetic polymers have also been evaluated as nonviral DNA vehicles. The principle is based on the concept of forming condensed DNA particles via complex formation with cationic polymers, referred to as polyplexes. The use of polycationic polymers leads to electrostatic neutralization of the anionic charges of DNA and subsequent condensation of the polynucleotide structure of DNA, thereby protecting it against nuclease digestion34,35. Furthermore, owing to the reduced dimensions of the molecules, transport of the compact polymer– DNA particles is facilitated through the extracellular matrix. As a result, the cellular uptake through endocytosis is enhanced. Many polycationic molecules have been used, including poly-L-lysine (PLL), polymethacrylate dendrimers, polyamidoamine and polyethyleneimine (PEI). Among these, PEI and PLL are the most common and important molecules used as nonviral vectors. PLL is a well-known polycation and has been used to deliver drugs for many years. It has also been used to condense pDNA under various salt conditions34,36–38. The resulting PLL–DNA particles have been shown to be protected against DNA degradation39,40. Electron microscopic studies have revealed that PLL–DNA complexes assume a rod-like appearance with a diameter of 15 nm and a length of 109 ± 36 nm and are thus much International Journal of Gerontology | June 2008 | Vol 2 | No 2

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200 nm 30 nm

Figure 1. Electron micrographs of poly-L-lysine–DNA (right) and partially condensed polyethyleneimine–DNA (left) complexes. With permission from reference 47.

smaller than lipoplexes (Figure 1). The poor circulatory half-lives of PLL–DNA complexes, typically shorter than 3 minutes, limit their use in vivo41–44. In general, PLL and PLL–DNA complexes have been reported to exhibit low immunogenicities41,45. Among the cationic polymers, PEI has been the most commonly used for gene delivery. Polycationic PEI is receiving much attention because of its characteristic of condensing DNA with an intrinsic endosomolytic activity46. Completely condensed PEI–DNA complexes are more homogenous and smaller in diameter than lipospermine (a cationic lipid)–DNA complexes (20–40 vs. 50–70 nm; Figure 1)47. The most prominent feature of PEI is its extremely high cationic charge density. Because every third atom of the PEI molecule is a nitrogen atom that can be protonated in the endosomal pH range48,49, PEI has the ability to capture protons that are pumped into endolysosomes, thereby acting as a proton sponge. This effect is presumably followed by passive chloride influx into the endosomes, which leads to osmotic swelling and disruption of the endosomes and permits the escape of the endocytosed PEI–DNA complexes. However, PEI is highly cytotoxic. Factors influencing its cytotoxicity include its molecular weight, incubation time, cation concentration and density of cationic groups50–52. Although the toxic effects of PEI on cells can be reduced by conjugation with other polymers, such as PEG53, this conjugation is not sufficient to completely solve the cytotoxicity problem.


Physical Approaches To date, there have been three major physical approaches to gene delivery, namely needle-free injection, electroporation, and US.

Needle-free injection Two devices have been developed that allow gene delivery by injection without needles. The first device, referred to as a “gene gun”54, uses a high-pressure helium stream to directly deliver DNA coated onto gold particles into the cytoplasm. The efficiency of the gene gun is variable, and the duration of the expression is transient. The relative advantages of the gene gun over some viral vectors are that it can be used to transfer genes into nondividing cells and the DNA–gold beads are cheap and easy to prepare. Gene gun delivery into the skin is a promising alternative to the injection of naked pDNA into muscle for genetic vaccinations55. The second device, called a “jet gun”, uses a DNAcontaining solution under high pressure for delivery into interstitial spaces. Jet injections of naked DNA may provide an option for keratinocyte gene therapy in the future56.

Electroporation Since 1982, the use of electric pulses for cell electroporation has been applied to introduce foreign DNA into prokaryotic and eukaryotic cells in vitro57. Electroporation uses electrical fields to create transient pores in 39

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C.H. Su et al

Propagation

Rarefaction compression

Amplitude

Pressure maximum Zero Pressure minimum

One cycle

Wavelength

Figure 2. Schematic representation of a continuous ultrasound wave. The peak ultrasound rarefaction pressure (pressure minimum) is proportional to the mechanical index.

the cell membrane that allow the entry of normally impermeable macromolecules into the cytoplasm. To date, electroporation has been used for in vivo studies of gene transfer into skeletal muscle21,58.

US-based Technologies in Gene Delivery US waves are defined as mechanical sound waves with frequencies above the audible sound range of humans, generally about 20 kHz59. The principle of piezoelectricity is commonly applied to generate US waves. Piezoelectric materials can be used as ultrasonic transducers for medical purposes. The application of a rapidly alternating potential across a piezoelectric crystal induces corresponding alternating dimensional changes, consequently converting electrical energy into sound waves. The direction of US wave propagation is the same as the direction of oscillation. The medium that the sound wave propagates through is alternately compressed (“compression” or “high pressure” zone; Figure 2) and stretched (“rarefaction” or “low pressure” zone; Figure 2), resulting in pressure variations in the medium.

Bioeffects of US The physical effects of US have been studied in vitro and in vivo, and they can be classified in two principal groups, i.e., thermal and mechanical. The mechanical group includes acoustic cavitation60, acoustic microstreaming61, and radiation pressure. Among these, acoustic cavitation is thought to be the most important bioeffect. Briefly, as the US waves propagate through the 40

medium, the characteristic compression and rarefaction cause microscopic gas bubbles in the tissue fluid to contract and expand, respectively. Two types of cavitation are recognized. Gas body activation62,63 and stable cavitation are terms used to describe bubbles that oscillate in diameter with the passing pressure variations of the sound wave. Generally, in gas body activation, only a relatively low level of US intensity is demanded to activate a preexisting gas body. Inertial cavitation63 or transient cavitation occurs when the bubble oscillations are so large that the bubbles finally implode violently, producing pressure discontinuities (shock waves), free radicals, extremely high localized temperatures (at least 5,000 K), pressures (up to 1,200 bars) and light (sonoluminescence).

Fundamental parameters of US The intensity of the US beam is one of the crucial parameters that determine the rate and extent of the thermal and nonthermal effects. Intensity (W/cm2) refers to the amount of energy contained in a wave as it passes through any one point. More recently, the mechanical index (MI)64,65 has come into use as an indicator or predictor of possible biological responses to cavitationrelated bioeffects. The MI is defined as: MI =

P f

where f is the driving frequency in MHz and P is the peak rarefactional (negative) pressure in MPa (Figure 2). P is the amount of negative acoustic pressure within a US field and is often used to describe the likelihood


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of causing a nucleus to undergo inertial cavitation in response to a series of US pulses.

Applications of US in gene delivery It is well known that ultrasound exposure (USE) can induce transient pore formation in the cell membrane66–69, referred to as sonoporation, which allows proteins and other macromolecules access into the cells. Sonoporation can be regarded as similar to the promotion of membrane permeability induced by US energy. Although researchers believe that nonthermal bioeffects (cavitation) play a crucial role in US-induced gene expression, the exact mechanism of which remains under investigation.

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LUC expression at 24 hours after USE was significantly increased by 2.4-fold with L1 and 1.7-fold with L2. The important results described above suggest that US can enhance gene delivery, possibly via cavitation, even without adding microbubble echo contrast agents (MECAs). In 2007, Deshpande et al.74 performed US-assisted in vitro reporter gene transfection and found that US sonication alone increased transfection by up to 18fold, while DNA complexed with PEI increased transfection by up to 90-fold, compared with a control DNA group. In addition, the combination of US and PEI synergistically increased transfection by up to 200-fold, resulting in a transfection efficiency of 34%.

MECAs and their applications in gene delivery In vitro applications of US in gene delivery Naked pDNA is the simplest nonviral vector. However, the phosphate groups on the deoxyribose rings of DNA confer a net negative charge on the molecule, thereby hampering its potential for electrostatic interaction with the anionic lipids in the cell membrane and causing a very low cellular uptake. Disadvantages for systemic gene delivery with naked DNA have also been found, because pDNA vectors can be rapidly degraded and neutralized by endogenous deoxyribonucleases. Therefore, it is reasonable to combine naked pDNA delivery with other methods to improve the transgene efficiency. In 1987, Fechheimer et al.70 first demonstrated that US had potential as a tool for pDNA delivery into murine fibroblasts. The first major investigation in this field came in 1996, when Kim et al.71 studied the potential use of USE as a novel transfection method for laboratory use. The maximal transfection rate was 2.4% of surviving primary chondrocytes, while ~50% of the exposed cells died. Lawrie et al.72 used a custom-built US transducer to expose cultures of porcine vascular smooth muscle cells (VSMCs) and ECs to very low intensity 1-MHz US (MI, 0.1; 0.4 W/cm2). USE for 1 minute was found to enhance luciferase transgene expression in porcine VSMCs by 7.5-fold and 2.4-fold at 48 hours posttransfection compared with naked plasmid transfection and lipofection, respectively. In 2005, Feril et al.73 investigated the effects of US (1 MHz) on liposome-mediated transfection, using three types of liposomes (L1, L2 and L3) containing dioleoylphosphatidylethanolamine and cholesterol at varying ratios. HeLa cells were treated with liposome (L1 or L2)–DNA complexes containing the LUC plasmid for 2 hours before USE (0.5 W/cm2; 1 MHz for 1 minute).


The concept of US contrast imaging was introduced in the 1960s and has significantly extended the use of US imaging during recent years owing to dramatic improvements in the stability, circulation time and echogenicity of MECAs. Because of their capability to increase the US backscatter signal from blood with minimal toxicity, MECAs have been applied in combination with conventional two-dimensional and Doppler imaging for diagnosing diseases and creating better images of the states of organs. The ideal MECAs should be nontoxic, intravenously injectable, capable of crossing the pulmonary capillary bed after a peripheral injection, and sufficiently stable to achieve enhancement for the duration of the examination. They are typically gas-encapsulated microbubbles of around 1–10 μm in diameter75,76. Contrast agents have a gas core that is filled with air or a higher molecular weight substance, such as perfluoropropane, with a lower aqueous solubility. The surrounding shell can be stiff (e.g., denatured albumin) or more flexible (lipids or phospholipids), and the shell thickness can vary from 10–200 nm. Microbubbles have been shown to lower the energy threshold for cavitation by US energy and to have the potential of enhancing cavitation77. When US interacts with MECAs, leading to cavitation, pDNA and fragments of the microbubbles are driven across cell membranes into the target cells78. Consequently, acoustic cavitation plays a critical role in USassisted gene delivery.

In vivo applications of US in gene delivery Recently, US gene delivery has been applied in several tumor cell lines, as well as in ECs and VSMCs72,79. In terms of transdermal delivery of various molecules 41

42

1 MHz/–

1 MHz/CW

1.3 MHz/CW

1 MHz/PW

Adult rat brain

Rat myocardium

Porcine saphenous vein graft (ex vivo)

1 MHz/–

Rat carotid artery

Rat carotid artery

1 MHz/–

Rabbit skeletal muscle

1.75 MHz/PW

2.2–4.4 MHz/CW

Porcine coronary artery (ex vivo)

Rat skeletal muscle

2 MHz/PW

Rabbit femoral artery

1 MHz/PW

1.3 MHz/PW

Rat myocardium

Mouse skeletal muscle

118 MHz/PW

Rat prostate tumor

2 MHz/–

1 MHz/CW

MC38 murine colon cancer

Rat kidney (ex vivo)

–/lithotripter shock wave

Frequency/ mode

–/1.8

–/1.5

5/–

2.5/–

–/1.9

3/–

2.5/–

–/–

2.5/–

–/1.2

50/–

–/1.5

0.3–833/0.01–0.46

20/–

–/–

Intensity (W/cm2)/ mechanical index

Summary of in vivo (or ex vivo) ultrasound-assisted transfections

Mouse melanoma

Model

Table 2.

Lumen and total vessel areas were significantly greater in the TIMI-3 group

6-fold compared with control


Led to a significant 50% reduction in intima/media ratio



Significant prolongation of graft survival

Led to a significant 50% reduction in intima/media ratio

Led to a significant increase in capillary density

Led to a significant increase in the expression of eNOS



10–15-fold compared with control



Enhancement

BR14

Lipid-stabilized MECA

Optison

Optison

DNA-loaded lipid-MECA

Optison

Optison

Optison

Optison

DNA-loaded albumin MECA

–

Albumin-coated gas-filled MECA

–

–

–

MECA

Akowuah et al.96

Bekeredjian et al.92

Shimamura et al.94

Hashiya et al.93

Christiansen et al.98

Lu et al.102

Azuma et al.95

Taniyama et al.69

Taniyama et al.68

Teupe et al.103

Amabile et al.100

Shohet et al.104

Huber et al.99

Manome et al.101

Miller et al.97

Authors

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1 MHz/PW Liver model in C57/BL6 male mice


MI = mechanical index; MECA = microbubble echo contrast agent; CW = continuous wave; PW = pulsed wave; eNOS = endothelial nitric oxide synthase; TIMI-3 = thrombolysis in myocardial infarction-3; GFP = green fluorescent protein; US = ultrasound; PLGA = poly(lactic-co-glycolic acid); PEI = polyethyleneimine.

Shen et al.87 Optison An 85-fold increase in reporter gene delivery (US group)

1.04 MHz/PW Prostate tumor model in nude mice

–

Chumakova et al.89 Air-filled PLGA/PEI/DNA nanoparticles

1 MHz/PW Thigh muscles of mice

–

An 8-fold increase of cell transfection efficacy in irradiated tumors

Sheyn et al.88 Optison Bone formation in the site of osteogenic gene (rhBMP-9) delivery

1 MHz/PW Rat subconjunctival tissue

5/–

Yamashita et al.86 Bubble liposome

1 MHz/PW Mouse femoral artery

1.2/–

Strong GFP staining noted in conjunctiva (US+bubble liposome group)

Inagaki et al.90

1.6 MHz/CW

1/–

Neointima/media areas were significantly reduced

MECA


Rat myocardium

–/1.6

Significantly increased capillary density

Lipid-stabilized MECA

Korpanty et al.91

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in vitro and in vivo, US has shown enhancing effects for in vitro and in vivo delivery of insulin80–82, glucose83,84 and heparin85. Although these are promising in vitro findings, USbased gene delivery is still in its infancy. Since 1996, there have been several in vivo investigations concerning US-assisted gene delivery with or without microbubbles. The in vivo studies of US-assisted gene delivery are summarized in Table 268,69,74,86–104.

Conclusion At present, there are two main reasons why gene therapy has not globally succeeded in the clinical setting, namely inefficient delivery of the gene of interest to its correct sites of action and safety concerns regarding some viral-based vectors that are 1–3 orders of magnitude more efficient than conventional nonviral techniques for gene delivery in vivo. Many gene delivery methods, such as liposome-mediated transfection, are much less efficient in vivo than in vitro. US has several potential advantages over other techniques, particularly in that it can be focused and therefore targeted toward specific and, if necessary, deep locations within the body. US gene delivery has been urged as an applicable tool because of its bioeffects, especially cavitation105. Last but not least, in efforts to further improve the level of transgene expression, targeted gene delivery may be one of the promising methods that will work through US. In this regard, it would be feasible to design targeted MECAs that can selectively bind to areas of interest in tissues or the body for either diagnostic or therapeutic purposes. These active targeting strategies can be achieved by the development of targeted microbubbles with antibodies or peptides attached to their shells106–109. As a result, targeted microbubbles with a specific ligand for the receptors expressed in the diseased area can be applied for either US imaging or potentially therapeutic purposes via US-induced cavitation. Theoretically, it should also be possible to load microbubbles with genetic material that is already condensed by polymers or liposomes or to modify the surface of the microbubbles. The important steps required to develop this technique will be to load ligand-modified MECAs with polymer/liposome-condensed genetic material (such as pDNA) without compromising their stability and to eliminate the cytotoxicity in vitro and in vivo. Such “smart” microbubbles may be applied as specific 43

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contrast agents for US to improve diagnosis, and also as therapeutic agents in US-based gene delivery in clinical settings.

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